PROJECT SUMMARY The receptor tyrosine kinase HER2 is amplified or overexpressed in approximately 25% of breast cancers and is associated with tumor aggressiveness and poor prognosis. While the FDA-approved therapy trastuzumab has been shown to have substantial clinical benefit, more than 60% of patients with HER2+ cancer do not respond to it, and nearly all others who initially respond inevitably develop acquired resistance, underscoring the existing unmet need for new therapeutic approaches for this disease. Recently, we identified a previously unanticipated metabolic vulnerability of HER2 breast cancers, including the ones resistant to trastuzumab, in overreliance on mitochondrial creatine kinase 1 (MtCK1) and by extension creatine phosphate shuttle for energy supply. We demonstrated that a known substrate analog cyclocreatine, converted by MtCK1 into phosphocyclocreatine that in turn acts as an inhibitor of creatine phosphate pathway, suppresses trastuzunab-resistant HER2 PDX tumor growth, thus providing proof of concept for small molecule therapeutic intervention. We hypothesize that pharmacological agents inhibiting MtCK1 will offer a novel much needed line of defense against HER2+ tumors as well as other tumors dependent on MtCK1 for survival such as EVI1+ AML. To test this hypothesis we propose to identify first-in-class small molecule inhibitors of MtCK1 using high-throughput large-scale screening (Aim 1), hit confirmation (Aim 2) and hit profiling and validation (Aim 3). The completion of our project will result in identification of compounds inhibiting creatine phosphate shuttle in HER2 cancers. We expect that the knowledge, developed strategies, and chemical probes will ultimately enable the development of innovative therapeutic approaches to combat HER2 breast and potentially other cancers.